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Light, Spectroscopy and the Spectrophotometer

Wavelengths of Light
Spectroscopy is the study of the interaction
between matter and radiated energy. In the
past, spectroscopy was basically the study
of how visible light dispersed according to its
wavelength by a prism. Later, the concept
was expanded to comprise any interaction
with radiative energy as a function of its
wavelength or frequency. Spectroscopic
data is often represented by - you guessed it
- a spectrum, which is a plot of the
response of interest as a function of
wavelength or frequency.



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The quantitative measurement of the absorption or transmission properties of a material

as a function of wavelength is called spectrophotometry, and the instrument we use is
called a spectrophotometer.
So were going to be looking at how light and matter interact. The nature of this
interaction depends on two things: the wavelength of the light (which we will control)
and the structure of the matter (which is what we are trying to study). Heres you new
best friend, the electromagnetic spectrum. Isnt it pretty?

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Ready, Set Choose Your Wavelength!

We want to use wavelengths which can excite electrons and move them to a higher
energy state. These wavelengths are in the visible / UV range, but even within that
range, we need to be more selective. It is important to note that molecules absorb only
those wavelengths of light that contain just the right amount of energy to move the
electrons to a higher energy state, resulting in peaks and valleys when
absorbance is plotted against wavelength.
Students often ask me what happens if you use longer or shorter
wavelengths (i.e., outside the UV / visible range). Well, you can - but youll
need different instrumentation and you would measure different properties
of matter. Absorption of longer, low energy wavelengths (infrared)
stretches and bends bonds in molecules; consequently IR spectroscopy is
used to identify chemical structures. Shorter wavelengths (X-rays and
gamma rays) are sufficiently energetic to strip electrons from molecules
completely. Wow.
Light vs. Matter - Quantified
You probably already know that when you
pass light through a substance, some of the
incident energy is absorbed and some is

The spectrophotometer measures transmitted light through a sample with respect to the
incident light very precisely. Check this out:

OK. As you can see, the spectrophotometer measures how much light is transmitted
through our sample (compared to how much light entered the sample). Transmittance
is simply the percentage of incident light reaching the photocell:
%T = 100 (I / Io)
where I is the light reaching the photocell and Io is the light striking the cuvette.

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Wait a second heres a graph of transmittance as a function of concentration:


As you can see, the relationship is exponential, which is awkward to work with. The
good news is that since transmittance decreases exponentially with concentration, log
1/T increases linearly with increasing concentration! Thats great because straight lines
are a lot more convenient than curved ones! Heres a graph of absorbance as a
function of concentration:

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The best news of all is that YOU dont have to do the math - the spectrophotometer
does it for you! All YOU have to do is tell the spectrophotometer that you want to know
absorbance (ABS) not %T. But just so you know A = - log %T
The relationship between absorbance and concentration is known as the Beer-Lambert law: A = elC ,
where e is the extinction coefficient (a property of the light-absorbing substance), l is the light path in cm
and C is the concentration of the light-absorbing substance. Most cuvettes have a light path of 1 cm. Just

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Using the Spectronic 20 Genesys Spectrophotometer


1. Check that the cell holder is empty.

2. Turn on the power switch at the back of the

unit and allow the spectrophotometer to warm up
for 30 minutes.

3. Press the A/T/C key to select ABS

(absorbance) mode. If you take your readings
in %T, YOULL have to do the math yourself later!!!

4. Set the wavelength to the desired value.

Holding the nm arrow keys will cause the
wavelength to change more quickly

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5. Wipe the cuvette with a kimwipe to remove

fingerprints. Holding the cuvette at the top,
insert the blank into the cell holder with the clear
walls of the cuvette to the front and back of the
instrument (if not using glass test tubes).
Remember - setting the blank is like hitting the TARE
button on a balance! The blank is used to zero readings
due to imperfections in the cuvette and for the
absorbance of light by the medium or carrier solution.

Close the sample compartment.

6. Press 0ABS/100%T to blank the
instrument. You will see 0.000 A in the
window (as well as the wavelength you


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7. Remove the blank from the cell holder. Mix contents of your sample if
necessary. Wipe fingerprints from your sample cuvette, insert your sample
and close the sample compartment.
8. Record the instrument reading in your lab notebook.
9. Remove the sample from the cell holder.
10. Say TA-DAH! Or do a hula. Your choice.